A highly efficient method for long-chain cDNA synthesis using trehalose and betaine

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• Full-length complementary DNA (cDNA) synthesis is crucial for various molecular biology techniques, including cDNA library construction and rapid amplification of cDNA ends (RACE-PCR).
• Secondary structures within RNA molecules can impede efficient reverse transcription, limiting the length and yield of synthesized cDNA.

Purpose of the Study:

• To investigate the effect of betaine and trehalose on the efficiency and length of cDNA synthesis during reverse transcription.
• To determine if combinations of betaine and trehalose can improve the yield of full-length cDNA products.

Main Methods:

• Reverse transcription reactions were performed with varying concentrations of betaine and trehalose.
• Real-time PCR was used to quantify the yield of synthesized cDNA, specifically targeting a 14-kb clarithrin mRNA template.
• The length of the synthesized cDNA products was analyzed.

Main Results:

• The inclusion of betaine alone and in combination with trehalose resulted in longer cDNA synthesis products.
• A combination of 2 M betaine and 0.6 M trehalose significantly enhanced cDNA yield, producing nearly 9 times more cDNA of 12.5 kb length from clarithrin mRNA.
• Betaine's ability to resolve RNA secondary structures and lower melting temperature was identified as the mechanism for improved cDNA synthesis.

Conclusions:

• Betaine and trehalose are effective additives for enhancing full-length cDNA synthesis.
• The combination of betaine and trehalose offers a significant improvement in cDNA yield and length, beneficial for molecular biology applications.
• This optimized reverse transcription method is recommended for all laboratory procedures requiring high-quality, full-length cDNA.